Paraquat is the prototype of a class of chemical agents which can transfer electrons from biological reductants to molecular oxygen within cells. By this action it can generate superoxide and hydrogen peroxide and deplete cells of reduced pyridine nucleotides. The effects of paraquat necessary for its toxicity in eukaryotes are, however, unknown. To determine how paraquat injures mammalian cells I propose to study cultured cells because of their homogeneity, of the ease of controlling experimental conditions, and of their suitability to a powerful genetic approach. If paraquat kills cells because it transfers electrons between NADPH and molecular oxygen, I should be able to demonstrate that these effects occur before cellular death. I therefore propose to correlate the effects of paraquat on cellular NADPH, on activity of the hexose monophosphate shunt, and on cyanide-resistant respiration with loss of cellular viability. If changes in all three occur before death of the cells, they are candidates for necessary steps in paraquat's toxic action. The mutagenicity of paraquat in bacteria raises questions about the delayed effects of paraquat in mammalian cells. I propose to assess whether paraquat can induce mutation and/or transformation in cultured cells, and the mechanism of this effect. The most powerful technique with which to understand the mechanism of the toxicity of paraquat is genetic. In cells genetically resistant to the toxicity of paraquat a necessary step in the toxic action of paraquat must have changed. Therefore if the adaptation can be identified and if it is the only change in the resistant cells, the mutation can define a necessary step in the toxicity of paraquat. For example, if paraquat must form superoxide anion to injure cells, one adaptation which would make cells resistant is an increased cellular content of the enzyme which catalytically scavenges superoxide, superoxide dismutase. I propose to characterize the clones of cultured cells made resistant to the toxicity of paraquat by repeated exposure to successive abrupt increments in concentration.